Deregulation of proliferation is a characteristic of tumorigenesis and therapeutic approaches for cancer treatment targets apoptosis, cell cycle arrest and differentiation. NAC has been shown to induce a multitude of molecular changes related to tumorigenesis . Recently, NAC has been demonstrated to inhibit apoptosis [5, 6], possess anti-inflammatory activities  and inhibit proliferation .
Here, we have monitored the reflection in global gene expression profiles of the transition from proliferation to a differentiated state in normal and cancer cells in vitro, as induced by NAC. Two out of three previous studies of the global gene expression that accompanies the spontaneous differentiation of Caco-2 report a general down-regulation of gene expression in differentiated cells as compared to the proliferating counterpart [9–11]. A similar, but not as pronounced, trend is reflected in the number of genes differentially expressed following NAC induced differentiation in Caco-2.
The expression level of 253 targets in Caco-2 and 414 in NHEK were statistically differentially expressed at different time points. Multiple appearances of differentially regulated transcripts were common, resulting in detection of totally less than 200 unique genes, respectively. This is fewer than expected in comparison to previous reports on differential regulation during spontaneous Caco-2 differentiation and probably due to the difference in stringency of the algorithms used for data analyses (MAS 4.0 vs RMA), rather than related to functional biological discrepancies.
In both data sets, the early responses were relatively limited and appeared to be transient, indicating that a large part of initial immediate early events occur at the level of translation and post-translational modifications. The nature of initial regulatory events is also expected to be transient due to feedback inhibition as well as to a restricted number of NAC induced mechanisms.
Interestingly, significant transcriptional down-regulation of the inhibitor of differentiation 1 (ID-1) was found at 1 hr in both Caco-2 and NHEK, suggesting a common mechanism of NAC induced differentiation and inhibition of proliferation in NHEK and Caco-2 epithelial cells. Analysis of ID-1 expression levels by real-time quantitative PCR confirmed the suppression of the transcript in both cell types (Table 3). ID-1 has been demonstrated to bind helix-loop helix transcription factors, preventing them from binding DNA . In particular, ID-1 has been shown to be required for G1 progression, and its constitutive expression inhibited the lineage commitment and differentiation in B-cells [13, 14]. Previous reports have also shown that ID-1 is a negative transcriptional regulator of CDKN2A (p16/p14/p19), which induces G1 arrest through the inhibition of Rb phosphorylation by cdk -4 and -6 . Overexpression of ID-1 was also reported in psoriatic involved skin . Inhibitors of histone deacetylase activity are emerging as a potentially important new class of anticancer agents. The cell cycle blockade and differentiation caused by such a drug, trichostatin A, caused decreased levels of ID-1 consistent with cell cycle senescence and differentiation of A2780 ovarian cancer cells . Vitamin D is also known to promote differentiation and was shown by others to down-regulate ID-1 through a suppressive vitamin D response sequence in the 5'of the gene . The ID-1 expression is regulated by a protein complex containing the immediate-early response gene EGR1 .
The growth regulatory properties of EGR1 have been found to involve coordinated regulation of TGF-β1 and fibronectin (FN1). The resulting proteins are secreted and lead to increased expression of plasminogen activator inhibitor-1 (PAI1). Both the secreted FN1 and PAI1 functions to enhance cell attachment and normal cell growth . We detect the induction of both fibronectin and PAI1 in NHEK cells at both 12 and 24 hrs, suggesting a role of EGR1 pathways in the NAC mediated mechanism at least in this cell type.
Other interesting down-regulated genes at 1 h after NAC treatment in NHEK included for example regulated in development and DNA damage response 1 (REDD1), squamous cell carcinoma antigen 1 (SCCA1), highly expressed in cancer (HEC), s100a9 and kallikrein 7 (also termed stratum corneum chymotryptic enzyme – SCCE). REDD1 has previously been shown to be down-regulated in differentiating primary human keratinocytes and ectopic expression inhibits in vitro differentiation . The suppression of SCCA1 has been demonstrated to inhibit tumour growth , HEC expression is increased in tumours  and S100A9 has been found to be up-regulated in psoriasis patients displaying keratinocyte hyperproliferation and altered differentiation . SCCE has been suggested to play a role in desquamation and its up-regulation is associated with poor prognosis of ovarian and breast cancer [25, 26]. A number of interesting up-regulated genes, such as activin A and WEE1, were also identified in NHEK 1 hr after NAC treatment. Over expression of WEE1 inhibits cell cycle progression by inactivation of the CDC2/cylin B complex , while activin A is a member of the TGF-β family of cytokines which is known to promote growth arrest and differentiation in several tissues including intestinal epithelia [28, 29]. In fact it was first identified as a protein that exhibits a potent differentiation-inducing activity . In Caco-2, the proto-oncogene fos and the transcription factor HNF3A, which have been observed to be amplified in human malignancies , was two of the identified genes being repressed at 1 hr after NAC treatment. A single gene, integrin alpha 2, was found up-regulated at this time-point.
It is clear that these data collectively describe molecular changes associated with the mediation of a differentiated epithelial phenotype. A large part of the differentially expressed genes have clear implications in withdrawal of mitogenic signals and in promotion of growth arrest. Multiple signalling pathways are suggested to be involved.
Late response (multiple occurrences)
Progressively more genes were affected in both cell types, and many showed similar trends in direction, over later time points. Cluster analysis revealed tightly linked genes between the 12 and 24 hrs time points in the same cell type and genes with such multiple appearances are potentially more strongly implicated in the differentiation process.
In NHEK cells, for example the down-regulated mitogens neuregulin 1 (heregulin) and melanoma growth stimulatory activity (MGSA) , belong to this group. MGSA belongs to a super family of chemochines, including IL-8, which is involved in inflammatory processes. Heregulin is known to activate the oncogenic ERBB2 receptor . Cdc-6, which are regulated in response to mitogenic signals, binds PCNA and is required for initiation of DNA replication , was also repressed at both 12 and 24 hrs after NAC treatment, implying programs involving withdrawal of mitogenic factors as important mechanisms for NAC mediated inhibition of proliferation and increased differentiation in NHEK cells. The expression of Topoisomerase II (TOP2) was also repressed, confirming results obtained in NAC treated CHO cells . Topoisomerases control and alter the topologic states of DNA, and the relaxation activity of TOP2 is essential for productive RNA synthesis on nucleosomal DNA .
The list of correspondingly important up-regulated genes in NHEK was extensive and included activin A, transglutaminase 2 (TGM2), ErbB3 (HER3), matrix metalloproteinase 9 (MMP-9), fibronectin (FN!), PAI1 and TGFβ among others. Notably, activin A was the only gene found to be up-regulated at all investigated time points, demonstrating a sustained growth inhibitory and differentiation promoting signal. TGM2 catalyses cross-linking of proteins, demonstrates G-protein function in receptor signalling  and was recently reported to phosphorylate IGFB3 . IGFB3 in turn has a major role in regulation of proliferation as a growth inhibitor through IGF2 binding and alternative IGF2 independent pathways . Thus implicating potential regulatory functions of TGM2 in proliferation and differentiation. Surprisingly, ErbB3, which promotes proliferation through the Wnt signalling pathway, was also up-regulated. RT-PCR could confirm the induction (Caco-2, 12 hrs) and a study investigating spontaneous Caco-2 differentiation is also in agreement with our data on up-regulation . In contrast, a recent publication reported its up-regulation in breast cancer , suggesting a dual role of ERBB3 in cell cycle regulation. The induction of MMP9, confirmed by RT-PCR (48 h NHEK) is in additional contrast to our observations of NAC-induced cell differentiation and proliferation. MMP9 has been associated with angiogenesis, tumour progression and metastasis as mediated through degradation of the extracellular matrix (ECM)  and stimulation of hyperproliferation . However, tumours with low levels of MMP9 were found to be less differentiated. Thus, although MMP9 stimulates proliferation, it is also implied in positive regulation of differentiation . NAC has been proposed to inhibit activation of latent MMP9 protein in ECM reservoirs by removal of its propeptide and by competing for the zink ion which is necessary for enzymatic function . Hence, our data may imply that post-transcriptional regulation of MMP9 prevails over the transcriptional changes as the major control mechanism.
A large number of multiple occurring differentially expressed transcripts were demonstrated in Caco-2, including intestinal trefoilfactor 3 (TFF3) and Aquaporin 3 (AQP3) among others. TFF3 has been shown to have a central role in the maintenance and repair of intestinal mucosa  and upregulation is expected during differentiation. Aquaporins (AQPs) are water channel proteins, important for the transport of water and other small proteins across the cell membrane . AQP1 has previously been shown to be involved in cell cycle control , suggesting that AQP3 may also have a role in the progression of cancer. AQP3 has been reported to be highly expressed in several types of stratified epithelial cells in rat, including the epidermis  and the differentiated cells of the gastrointestinal tract . The expression of AQP3 was reported to be up-regulated in differentiating Caco-2 cells , while expression was shown to be down-regulated in differentiating primary keratinocytes . In this study AQP appears to have a transient behaviour in Caco-2 cells with a repressed behaviour at initial time point and induced pattern at later time points (confirmed by RT-PCR), indicating a remodelling of cell membrane constituents.
The genes repressed in Caco-2 included for example Cyclin D1, Inhibin beta B, BMP-2 and FHL-2. The D1 cyclin is involved in β-catenin-TCF signalling and its down-regulation induce G1 arrest . FHL-2 has been demonstrated to be a coactivator of β-catenin from cyclin D and IL-8 promoters in a colon cell line , suggesting that repression of FHL-2 may also repress growth. Inhibin beta and BMP-2 are members of the TGF family of genes. Inhibin beta is an antagonist of activin A activity and consequently represses differentiation and promotes growth . BMP-2 on the other hand, has been demonstrated both to induce apoptosis  and growth inhibition/differentiation . In contrast, another recent study demonstrated the ability of BMP-2 to enhance the growth of tumours .
Late response (single occurrence)
A vast number of additional interesting genes with potentially important roles in mediation and manifestation of the differentiated epithelial phenotype was identified as significantly induced or repressed in a single time point.
As an example, up-regulation in NHEK after 24 hrs was seen for transcripts encoding CDKN2B (p15), which is believed to be an effector of TGF-β induced G1 arrest and inhibition of proliferation , and for CDKN1C, which is a p21 homologue and negative regulator of cell proliferation . Other up-regulated genes at this time point were the transcription factor Jun and BTG1. Jun was recently demonstrated to be a regulator of erythroid differentiation  and Jun B knock out mice have been shown to develop a proliferative disease resembeling human chronic myeloid leukemia . BTG1 has been proposed to belong to a family of antiproliferative genes . Up-regulation was also found for cadherin 13, a gene with growth inhibitory functions that is expressed in normal cells but not in the majority of human tumour cells of epithelial origin . While not identified by global transcript analysis, RT-PCR investigation revealed increased levels of E-cadherin in both Caco-2 (24 hrs) and NHEK (24 and 48 hrs), this in agreement with the previously reported immunohistochemical data  that showed increased staining of E-cadherin in NAC treated cells. In Caco-2 cells, the differentiation-related gene NDRG1, which is expressed during differentiation and down-regulated in colorectal neoplasms , was up-regulated at 12 hrs. This increase was also demonstrated by RT-PCR at 12, 24 and 48 hrs after NAC treatment. The induction of Cdx2 may also be a functional change, since reduced expression of Cdx2 has been shown to be important in colon tumorigenesis . Interestingly, in correlation with the controversial results from NHEK, an up-regulation of ErbB3 was identified in Caco-2 at 12 hrs. In addition, the oncogene myc was also up-regulated in contrast to the expected decrease. The down-regulation of Cox-2 and BMP-2 was also in concordance with NHEK data. In addition, the TGF-β family member BMP-4 was repressed. This correlates well with our results on repressed ID-1 expression, since both BMP-2 and -4 up-regulate ID-1 [64, 65].
Hence, although induced by the same mechanism (NAC) and yielding the same end-stage of growth inhibition and differentiation, the processes in NHEK and Caco-2 are on the whole quite different. This is demonstrated by gene specific differences that result in lack of correlation between cell types at the same time point after treatment as identified by cluster analysis. This is furthermore supported by the Gene Ontology analysis, tables 4 and 5, indicating that the two cell lines achieve their differentiated states using two distinct mechanisms, this in concordance with previously observed effects of NAC treatment on cell morphology and growth arrest . It should be noted that apoptosis does not appear to act as a regulating mechanism, since only a very small proportion of apoptotic genes are affected in either direction (2 to 12 genes out of 317, in either direction). This is also supported by the previous study , which analysed apoptosis by propidium iodide labelling and flow cytometry.
As additional testimony to the lineage specific differentiation programs, only very few genes were identified as being similarly regulated in both cell-types. These included ID-1, AQP3 and ErbB3 (as described above). Cox-2 was also similarly down-regulated in both cell types after NAC treatment. When investigating Cox-2 expression by RT-PCR we could confirm the down-regulation at 24 hrs in Caco-2 and identify an additional repression in NHEK at 1, 3 and 48 hrs after NAC treatment. Overexpression of Cox-2 has been shown to promote cell migration and invasion in Caco-2 cells  and to regulate colon carcinoma induced angiogenesis by production of angiogenic factors . In addition, epidermal differentiation is also affected by Cox-2 over expression. Cox-2 seems to prevent entrance into the postmitotic state, which is coupled to the switching on expression of differentiation-associated proteins, allowing keratinocytes to proliferate . In correlation with our data, a NAC mediated inhibition of Cox-2 expression have previously been demonstrated in colorectal cancer . Hence, it is likely that Cox-2 repression is a NAC specific event endorsing differentiation/growth arrest in both NHEK and Caco-2. HBP1 was also up-regulated in both Caco-2 and NHEK after 12 h of NAC treatment and RT-PCR could confirm an increase in Caco-2 at 12 and 24 hrs. This corresponds to previous findings, where HBP1 has been seen to have a negative effect on tumours. It has previously been established that HBP1 is a target of the retinoblastoma pathways [70, 71] and that HBP1 negatively regulates Wnt/β-catenin, thus inhibiting proliferation and suggesting that HBP1 may have a tumour suppressor function . Two additional proteins, putative 28 kDa protein and proline rich nuclear receptor coactivator-1 (PNRC1), were also identified as differentially expressed in both cell types. RT-PCR analysis was able to confirm an up-regulation of PNRC1 in Caco-2 24 hrs after treatment. However, these genes are not previously described and potential functions remain unresolved.
A proportion of the differentially expressed transcripts were not possible to predict as being part of the differentiation context. For example, ErbB3, fos, TGFβI and myc were found to be expressed at higher levels in differentiated cells, in contrast to their roles in promotion of proliferation. Interestingly, a similar contrasting increase in fos and ErbB3 levels was found in normal colonic cells as compared to colorectal cancers in a SAGE study . It is important to note that spontaneous morphological and functional differentiation in Caco-2 have been demonstrated not to be coupled, with independent mosaic patterns of proliferating and differentiated cells present adjacent in the cell culture , which may in part explain some of the contrasting results. However, many of the detected differentially expressed genes in this study have previously been described as altered in differentiated epithelial cells. In NHEK cells we could for example confirm the expected increased expression of SPRR1B and SPRR2A. In Caco-2 the up-regulation of AQP3, NDRG1 and TFF3 were among the genes that validated the results from the global transcription profile analysis.
Interestingly, several genes with major relevance in psoriasis have been found differentially expressed as a consequence of NAC treatment in the particular epithelial cells included in this study, for example S100A9, ID-1 and Cox-2. These findings could give a mechanistic background to the ongoing clinical studies being carried out based on empirical NAC treatment of patients having psoriasis.
Alternative NAC signalling mechanisms at the level of proteins and metabolites may also be important. For instance the phospholipid modulator, platelet activating factor, has been demonstrated to induce differentiation and inhibit proliferation in colon cells , and inhibit proliferation in cultured human keratinocytes . Accordingly, we are performing supplementary proteome and metabolome studies. In addition, analyses of additional cell lines for finding a common pathway of molecular changes that result from NAC induced differentiation are being considered.